While the
zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from
ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of
NADPH oxidase (NOX)
isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor
p67phox. Mouse hearts or H9c2 cells were subjected to I/R.
Protein expression was detected with Western blotting.
Infarct size was measured with TTC staining. The cardiac-specific
p67phox conditional knockout mice (
p67phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger
N-acetylcysteine (NAC) and the NOX inhibitor
diphenyleneiodonium (DPI).
p67phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of
p67phox by
siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of
p67phox, whereas the STAT3 inhibitor
stattic abrogated upregulation ZIP2 expression, indicating that the increase of
p67phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn2+ markedly enhanced
p67phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn2+ had the opposite effects, indicating that cardiac Zn2+ loss upon reperfusion triggers
p67phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via
p67phox, and cKO of
p67phox aggravated cardiac injury after I/R, indicating that
p67phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of
p67phox expression in response to Zn2+ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3.